Highly parallel microbial diagnostics using oligonucleotide microarrays

wwwelseviercomlocateclinchim

Clinica Chimica Acta 36

Review

Highly parallel microbial diagnostics using oligonucleotide microarrays

Alexander Loy a1 Levente Bodrossy b

a Department of Microbial Ecology University of Vienna A-1090 Vienna Austriab Department of BioresourcesMicrobiology ARC Seibersdorf research GmbH A-2444 Seibersdorf Austria

Received 3 April 2005 accepted 5 May 2005

Available online 26 August 2005

Abstract

Oligonucleotide microarrays are highly parallel hybridization platforms allowing rapid and simultaneous identification of many

different microorganisms and viruses in a single assay In the past few years researchers have been confronted with a dramatic increase in

the number of studies reporting development andor improvement of oligonucleotide microarrays for microbial diagnostics but use of the

technology in routine diagnostics is still constrained by a variety of factors Careful development of microarray essentials (such as

oligonucleotide probes protocols for target preparation and hybridization etc) combined with extensive performance testing are thus

mandatory requirements for the maturation of diagnostic microarrays from fancy technological gimmicks to robust and routinely

applicable tools

D 2005 Elsevier BV All rights reserved

Keywords Microbial diagnostics Microarray Hybridization Oligonucleotide Marker gene

Contents

106

110

110

110

111

114

114

114

1 Basic concept

2 Microarray hybridization formats

3 Development and analytical performance

31 Resolution choice of marker genes and probe lengths

32 Development and optimisation of microarray probe sets specificity sensitivity and uniformity

33 Further selected strategies to increase specificity andor sensitivity

4 Data analysis and quantification potential

5 Diagnostic applications

6 Conclusions and outlook

115

Acknowledgements 116

References 116

1 Basic concept

DNA microarray (microchip biochip gene chip) tech-

nology allows the parallel analysis of highly complex gene

0009-8981$ - see front matter D 2005 Elsevier BV All rights reserved

doi101016jcccn200505041

Corresponding author Tel +43 50550 3548 fax +43 50550 3444

E-mail addresses loymicrobial-ecologynet (A Loy)

LeventeBodrossyarcsacat (L Bodrossy)

URLs wwwmicrobioal-ecologynet (A Loy) wwwarcsacatuub

microbiology (L Bodrossy)1 Tel +43 1 4277 54207 fax +43 1 4277 54389

mixtures in a single assay and thus symbolises as no other

method the (post-)genomic era of high-throughput science

While microarrays initially emerged as tools for genome-

wide expression analysis and are nowadays routinely used

for this purpose they are also increasingly being developed

for diagnostic applications [1] Microbial diagnostic micro-

arrays (MDMs) consist of nucleic acid probe sets with each

probe being specific for a given strain subspecies species

genus or higher taxon [1] A characteristic MDM experi-

ment is depicted in Fig 1 MDMs fall into two distinct

3 (2006) 106 ndash 119

Fig 1 Typical steps of a diagnostic microarray experiment

A Loy L Bodrossy Clinica Chimica Acta 363 (2006) 106ndash119 107

categories according to their intended use Environmental

MDMs [23] are primarily applied in environmental and

industrial microbiology to obtain a picture of the structure of

the microbial community being analysed Requirements for

this class of MDMs are the parallel detection of many

microorganisms at the level of species genus or a higher

taxon and the potential for some level of quantification

Detectionidentification MDMs typically applied in clin-

ical veterinary food and biodefense microbiology [45]

must usually enable the reliable detection andor identifica-

tion at the speciessubspeciesstrain level of one or a few

microbes out of many that may be present in a sample It

Table 1

Selected applications of oligonucleotide microarrays for microbial diagnostics

Platform Target organisms Marker gene(s) Highest

phylogenetic

resolution

L 1) No of probes Field 2) No of

samples

analysed

Sample type Reference

Planar glass slide Entamoeba histolytica

E dispar Giardia lamblia

Cryptosporidium parvum

Various Species subtypes 20ndash30 88 CF ndash ndash [79]

Planar glass slide Cryptosporidium hsp70 Isolates 15 68 E ndash ndash [94]

Affymetrix Bacteria 16S rRNA Higher level

bacterial taxa

20 31179 E 1 Air filtrate [17]

Planar glass slide Bacteria involved in

nitrification denitrification

nitrogen fixation methane

oxidation and sulfite reduction

nirS nirK amoA nifH

pmoA dsrAB

Species 50 763 E 1 Marine sediment [47]

Planar glass slide 3) Bacteria causing abortion and

sterility in mares

23S rRNA Species subspecies 24ndash32 32 C 21 Cervical swabs [21]

Planar glass slide Bacterial fish pathogens 16S rRNA Species 22ndash31 18 CF ndash ndash [18]

Planar glass slide 4) Bacterial fish pathogens cyt rpoN gyrB toxR ureC

dly vapA fatA A plassal

Subspecies 25 9 EF ndash ndash [95]

Planar glass slide Campylobacter jejuni C coli

C lari C upsaliensis

fur glyA cdtABC ceuBC fliY Species 17ndash35 74 C 16+6 Isolates and

mixed cultures

[76]

Planar glass slide 4) Campylobacter spp 16S rRNA 16Sndash23S

intergenic spacer

Campylobacter-specific genes

Species 27ndash35 5 CF 10+65 Chicken cloacal

swabs

[9697]

Planar glass slide 5) Cyanobacteria 16S rRNA Above genus 20ndash29 19 E 1 Lake water [98]

Planar glass slide Enterococcus 16S and 23S rRNA Species 41 18 CF 2 Milk [99]

Planar glass slide Escherichia coli Shigella Sal-

monella

gyrB Species 15ndash19 10 C ndash ndash [27]

Planar glass slide Selected taxa of marine bacter-

ioplankton

16S rRNA Higher taxa 15ndash20 21 E 1 Sea water [58]

Planar glass slide Listeria iap hly inlB plcA plcB clpE Species 17ndash33 132 C ndash ndash [36]

Planar glass slide Listeria spp Campylobacter

spp Staphylococcus aureus

Clostridium perfringens

Various Species 17ndash35 178 BF ndash ndash [100]

Planar glass slide Marine bacterioplankton 16S rRNA Species 15ndash20 21 E 1 Sea water [101]

Planar glass slide Methanotrophs pmoA Species subspecies 17ndash27 61 E gt100 Landfill cover soil [139]

Planar glass slide Mycobacterium spp gyrB Species 13ndash15 28 C 40 Human sputum [102]

Planar glass slide 6) Rifampin-resistant

Mycobacterium tuberculosis

rpoB Strain 15ndash16 11) 18 C ndash ndash [103]

3D-surface Rifampin-resistant


rpoB Strain 15ndash23 43 C 31 Clinical samples [90]

Planar glass slide 4) Pathogenic Vibrio spp vvh viuB ompU toxR tcpI

hlyA tlh tdh trh etc

Species 30ndash32 13 CF 30 Oyster [82]

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icaChimica

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363(2006)106ndash119

108

Planar glass slide 7) Quinolone-resistant

Escherichia coli

gyrA Genotype 19 42 C ndash ndash [104]

Planar glass slide Respiratory bacterial pathogens gyrB parE Species 20ndash24 27 C 94 Middle ear fluid

throat swabs

[105]

Planar glass slide Rhodocyclales 16S rRNA Species 18 79 E 1 Activated sludge [56]

3D-surface Some Bacillus spp 16S rRNA Species 15ndash23 30 BC ndash ndash [64]

Planar glass slide 7) Some Bacillus spp 16Sndash23S intergenic spacer Species 18ndash22 42 BC ndash ndash [22]

Planar glass slide Sulphate-reducing prokaryotes 16S rRNA Species 18 132 CE 6+8 Hypersaline

cyanobacterial

lake mat periodontal

tooth pocket fen soil

[15106]

3D-surface Thermophilic anaerobic

Archaea and Bacteria

16S rRNA Genus 17ndash20 17 E 3 Oil reservoir

formation waters

[77]

Affymetrix 8) Major potential biowarfare agents (bacteria viruses

eukaryotes)

Various Species 20 53660 BC 2 Air filtrate [107]

Planar glass slide Acute respiratory

disease-associated

Adenoviruses

E1A fibre hexon Serotypes 60ndash72 36 C 19 Throat swabs nasal

wash samples

[80]

Planar glass slide Human group A rotaviruses VP7 Genotype 18ndash26 50 C ndash ndash [108]

Planar glass slide Human papillomaviruses E1 Types 20 51 C 130 Clinical samples [109]

Planar glass slide 5) Human papillomaviruses Various Types 14ndash28 10 C 100 Clinical samples [110]

Planar glass slide Human papillomaviruses Various Types 30 27 C 73 Clinical samples

(tonsillar cancer)

[111]

Planar glass slide Influenza viruses Various Subtype 17ndash29 476 C ndash ndash [112]

3D-surface 9) Influenza viruses Various (ao hemagglutinin

and neuraminidase genes)

Subtypes 45ndash65 29 C ndash ndash [113]

Planar glass slide Orthopoxviruses C23LB29R gene ORF 62

(varicella-zoster)

Types 13ndash21 57 BC ndash ndash [114]

3D-surface Orthopoxviruses crmB Species 12ndash16 15 C ndash ndash [115]

Planar glass slide 10) Viruses Various Serotype 70 1600 C 6 Clinical samples [50]

1) Length of probe [nt]2) B biodefense C clinical microbiology F food microbiology E environmental microbiology3) On-Chip PCR4) Multiplex PCR6) DNA ligation detection reaction5) SOLAC-short oligo ligation assay on chip8) Perfect match and single mismatch probes used7) 3ndash10 diagnostic regions were identified per microorganism and each one of them was covered by 100ndash300 probes9) Flow-Thru Chip10) 70mer discriminatory oligonucleotides were designed based on all published viral genome sequences11) Probes comprised of 5 specific nucleotides (participating in specific ligation)+10ndash11 spacer T residues

ALoyLBodrossy

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icaChimica

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A Loy L Bodrossy Clinica Chimica Acta 363 (2006) 106ndash119110

should be noted however that there are also medical

microbiology contexts where (as described for environ-

mental MDMs) the microbial community structure has to be

addressed eg the composition of the human gastro-

intestinal tract microbiota

Although recent studies (Table 1) undoubtedly demon-

strate that MDMs are valuable tools for identification of

microorganisms and viruses in a highly parallel fashion

their use for routine diagnostics is still hampered by a lack

of standardisation (regarding factors such as probes target

genes hybridization platforms protocols and data analy-

sis) and insufficient evaluation of newly developed

MDMs

In this review we summarise recent progress in the

MDM field Many (seemingly conflicting) parameters and

steps have to be integrated during microarray design and

application in order to fulfil the potential of MDMs as

high-throughput screening tools for routine diagnostic

purposes We thus highlight crucial points related to

development and evaluation of MDMs The key to a

validated MDM and consequently to reliable results is

rigorous in silico and in vitro performance testing

Furthermore we exemplarily show recent technological

advances which should further improve the use of MDMs

in the near future


Although many diverse microarray platforms have

become available over the past few years [6] only a limited

number of solid supports are currently used for MDMs For

Affymetrix microarrays (wwwaffymetrixcom) oligonu-

cleotide probes are synthesised directly onto the microarray

surface by employing specific masks and the photolithog-

raphy method This approach enables a very high probe

density (well over 100000 probes per microarray) The

high price low flexibility and lack of a suitably high

number of validated oligonucleotide probes currently limit

the wide application of Affymetrix GeneChips in microbial

diagnostics In contrast the NimbleGen technology

(wwwnimblegencom) uses digital micromirrors instead of

physical masks to guide on-chip probe synthesis allowing a

more flexible design of custom-made high-density micro-

arrays Specific three-dimensional microarray formats such

as the gel-pad platform (wwwbiochipruen) [78] or the

PamGene system (wwwpamgenecom) [910] are coupled

with appropriate hybridization and detection devices that

offer the option to record hybridization and dissociation

events in real-time Melting curves for all probes on a

microarray can thus be rapidly established making the

development of validated probe sets significantly easier

[1112] However these systems are currently available

only in a few laboratories

The pioneer microarray format and still the most widely

used miniaturised solid support for the covalent immobili-

sation of probes are planar 13 in glass slides Oligonu-

cleotides are in most cases tethered via their 5V ends to

reactive groups on the coating layer of the glass surface The

establishment of microarray core facilities (including micro-

array spotting and detection devices) in many laboratories

and the general utility flexibility and moderate price of

planar glass microarrays are mainly responsible for the

success of this standard format



Two main parameters affect the resolution of a diagnostic

microarray assay (i) the degree of conservation of the

marker gene and (ii) the length of the oligonucleotide used

as a probe to target it [13]

The most widely employed target molecule for the

detection and phylogenetic analysis of microorganisms is

the small-subunit ribosomal RNA (SSU rRNA) and its gene

[714ndash18] The popularity of the SSU rRNA is reflected in

the existence of large and regularly updated sequence [19

20] and probe databases (httpwwwmicrobial-ecologynet

probebase) [16] for this target molecule The main

limitation of using the SSU rRNA (gene) as a marker in

microarray assays is that resolution below the species level is

generally not possible owing to high overall sequence

conservation Because differentiation of strains is often

essential in clinical diagnostics in order to initiate appro-

priate treatment of an infection less-conserved target

molecules are needed Potential probe targets which offer

strain-level resolution include (i) the large-subunit ribosomal

RNA (LSU rRNA) [21] (ii) the SSU-LSU rRNA intergenic

spacer region [22] (iii) house-keeping genes (eg rpoB

[23ndash25] gyrA [26] gyrB [27] recA [28] tuf [2930] groEL

[31] atpD [30] ompA gapA pgi [32] tmRNA [33]) (iv)

virulence genes [34ndash37] (v) antibiotic resistance genes

[938] (vi) functional genes encoding enzymes responsible

for specific metabolic traits [139ndash42] etc (for a detailed

list please refer to wwwarcsacatuubmicrobiology)

However individual sequence databases for these alternative

markers if they exist at all currently contain considerably

fewer entries than the SSU rRNA databases constraining the

development and evaluation of encompassing probe sets for

microarrays [43]

The hybridization properties of long oligonucleotide

probes (typically 50ndash100mer) which show pronounced

hysteresis (higher temperatures for dissociation than for

association) are fundamentally different from those of short

probes (typically 15ndash30mer) [4445] While in principle

short oligonucleotides allow the discrimination of single

nucleotide differences under optimal conditions (see below

for further details) this does not hold true for long

oligonucleotides Their threshold for differentiation is

approximately 75ndash87 sequence similarity [414647]


However probes of increased length display orders of

magnitude higher target binding capacities and hence the

use of long oligonucleotides should generally improve the

detection sensitivity of a microarray [48] Typically long

oligonucleotide MDMs are used in combination with

universal (not PCR based low bias) amplification strategies

or without any amplification [474950] In these approaches

probes target various non-conserved genes which are

specific to the microorganisms targeted Thus the low

differentiation power of long oligonucleotide probes is

compensated for by the host specificity of the genes they are

designed against With or without universal amplification

the resulting target represents the entire gene pool present in

the investigated sample without any reduction in its

complexity Higher binding capacities of the long oligop-

robes and higher target complexity result in similar relative

detection thresholds to that of short oligoprobes in

conjunction with PCR amplification (approximately 5)

[14751]

There is no universal answer to the question of which

probe-target combination is the best for a diagnostic

microarray as this will depend strongly on the intended

application In principle the highly parallel nature of

microarrays allows various probes of different length and

targeting different genes to be applied simultaneously

although homogenous hybridization behaviour of complex

probetarget combinations remains a problem (see below)

32 Development and optimisation of microarray probe

sets specificity sensitivity and uniformity

A crucial and challenging first step in the development of

oligonucleotide microarrays is the design of a suitable set of

probes and this is thus presented in more detail in the

following section

The following criteria set the quality standard for a

microarray probe set All probes on a microarray should

(i) be highly specific for their target genes ie not cross-

hybridize with non-target sequences (specificity) (ii) bind

efficiently to target sequences to allow the detection of

low abundance targets in complex mixtures (sensitivity)

and (iii) display a similar hybridization behaviour ie

similar thermodynamic characteristics under the same

experimental conditions (homogeneity uniformity)

Unfortunately these ideals represent conflicting goals in

practice and thus efforts to fulfil these criteria need to be

carefully balanced during probe design and experimental

procedures

Design of microarray probes in silico usually entails the

use of specific software tools in conjunction with an

underlying sequence database Numerous software tools

have been developed and widely applied for the parallel

design of oligonucleotide probes for whole genome

microarrays [5253] but not so for diagnostic microarrays

Although only one probe can be designed at a time

making the design of multiple probes laborious the probe

software tool of the ARB program package [19] is

commonly used for the design of 10ndash100mer oligonucleo-

tide probes for diverse hybridization formats [155455]

including diagnostic microarrays [56ndash58] The first step in

the design of a diagnostic oligonucleotide is selection of

the target group either arbitrarily or based on sequence

analysis (eg a phylogenetically coherent group of

organisms) (Fig 2) Second depending on user-defined

settings the ARB probe search algorithm identifies unique

sequence stretches which could serve as probe target sites

and subsequently returns a ranked list of potential

oligonucleotides Third the suggested probes can be

matched against all (usually aligned) sequences in the

database This probe match option of ARB is highly

beneficial because the user has the opportunity to evaluate

the specificity of a given probe in silico by checking for

the number types and positions of mismatches to non-

target sequences It is vital to stress that the value of this

evaluation step and thus the quality of a developed probe

set will strongly correlate with the completeness and up-to-

dateness of the underlying sequence database Regarding

the position of mismatches to non-target organisms a rule

of thumb for short oligonucleotides is that mismatches

located in the middle of the probe-binding site destabilize

more strongly the formation of the probe-target duplex In

contrast an even distribution of mismatches in the probe-

binding site is required to achieve optimal discrimination

for long oligonucleotides [4648] Please note that single

mismatches located at the terminal or next-to-terminal

position can hardly be resolved in microarray hybridization

[114857] In a further step the local alignments of

perfectly-matched and mismatched target sites can be used

as input for other freely-available software such as

CalcOligo (wwwarcsacatuubmicrobiology) Mfold

[59] or HyTher [60] to calculate the theoretical thermo-

dynamic properties of these potential hybridization events

However these nearest-neighbour algorithms for the

calculation of free energies (DG) and melting temperatures

(Tm) have been developed for hybridizations in solution

involving known concentrations of probe and target

molecules factors which are not readily known or do

not apply for hybridization of immobilized probes on

microarrays Data obtained in the laboratory must therefore

accurately verify the use of these algorithms to predict

potential cross-hybridization events of a microarray probe

[1153961]

For single probes it is in most cases possible to adjust

experimental conditions in such a way that no cross-

hybridization occurs However the most widely used

microarray hybridization formats only allow hybridization

andor washing at a single stringency making it impossible

to provide optimal hybridization conditions for all probes

on a microarray Thus promiscuous binding of some

probes to non-target sequences (false-positive signals) is a

frequently encountered problem [115] Nonetheless sev-

eral approaches still guarantee the reliability of identifica-

Creationupdate ofsequence database

Probe design

Specificity sensitivity homogeneity

Secondary structures (∆G Tm)

Predicted hybridization behavior

Thorough experimental evaluation(Specificity sensitivity homogeneity)

Pre-selected probe set

Refined probe set

Elimination of ldquobadrdquo probes

Suitable software

Identification of target groupeg by phylogenetic analysis

Fully evaluatedfinal microarray

Re-design

Preliminary microarray

In silico

In vitro

Type number position of mismatch(es)

Re-evaluation

12

34

ARB

ARB

ARBPrimRose

ARBPrimRoseOligoCheckCalcOligoMfoldHyTher

CalcOligoMfoldHyTher

Fig 2 Schematic representation of the procedures involved in the development and evaluation of a microarray probe set Types of molecular interactions of the

probe and target molecules 1 oligo-oligo dimer 2 oligo hairpin 3 secondary structure of the target 4 oligo-target duplex Free energies and melting

temperatures of the different types of interactions of probe and target molecules can be calculated to estimate their influence on duplex yield


tion by microarrays One approach involves the design of

multiple probes having identical specificities for the same

target sequence or group of sequences Therefore all

probes in a set of probes perfectly matching the target

organism must show positive hybridization signals to

minimize the risk of false-positive identification

[346263] This multiple probe concept is further extend-

able by probes having hierarchical (nested) specificities for

the target sequences (Fig 3) which also enables the

detection of novel members of known groups [54] Nested

probes can be best exploited if rRNA or its gene serves as

the target molecule [15545664] In contrast the highly

variable third codon (lsquolsquowobblersquorsquo) position in protein-coding

target genes hampers the design of probes having a broader

specificity and spanning more distantly related sequences

In addition to or instead of the use of multiple probes for

one marker gene it is possible to use more than one marker

gene of a target organism as a probe target during

hybridization This multiple probe-multiple target strategy

additionally increases redundancy and hence the confi-

12345678910111213141516171819202122232425262728

A1 A2 A3

B1 B2C1

D1

E1

A4

Phylogeny Sequence alignment

Fig 3 Illustration of the nested multiple probe concept A schematic sequence alignment is displayed in dashed lines Colored boxes show perfectly matched

probe target sites As an example sequence number one is unambiguously rated as present if each probe in the set of probes E1YD1YC1YB1 B2YA1

A2 A3 perfectly matching this sequence at identical and hierarchical levels of specificity shows a positive hybridization signal


dence in a positive call [6263] Furthermore careful

selection of multiple marker genes may also allow

genotyping of the detected microbes Information on

virulence factors antibiotic resistance etc can thereby

be obtained simultaneously in a single diagnostic assay

Another prominent way (also applied in the Affymetrix

GeneChips protocols [17]) to ensure specificity is the

inclusion of so-called mismatch-control probes on the

microarray [6566] Comparison of signal intensities from

perfectly matched and mismatched probes allows cross-

hybridization to be identified and its extent estimated

In summary one set of experimental conditions is not

suitable to ensure absolute specificity of all probes during

simultaneous hybridization on a microarray However

microarrays offer possibilities to compensate for lack of

specificity on the single probe level by inclusion of a

multiplicity of redundant probes an option which is not

available for traditional hybridization formats

Sensitivity is (i) commonly defined as the lowest

absolute andor relative abundance of the target group

which is detectable and (ii) dependent on the duplex yields

of the individual probes on a microarray To avoid the

worst-case scenario low sensitivity of a given probe

leading to a false-negative result a variety of parameters

can be adjusted during the design of the actual microarray

and the experimental set-up An important strategy for

increasing duplex yield is to reduce steric hindrance during

microarray hybridization Lifting the oligonucleotide probe

physically away from the surface of the microarray by the

introduction of simple spacer elements enhances its

accessibility by several orders of magnitude [6768]

Furthermore the tendency of a probe and its target to form

stable secondary self-structures must be minimized

[16970] Tm and DG of secondary structures can be

calculated by using the software tools mentioned above but

should be treated cautiously due to the limited knowledge

on the thermodynamics of microarray hybridization (see

above) If RNA is the target molecule complexity of the

target is typically reduced by physical or chemical

fragmentation [14571] A popular method to create short

DNA fragments of varying length is by random prime

labelling [49] Partial digestion with DNase I [49] or use of

hydroxyl radical-producing reagents are other options to

fragment DNA the latter non-enzymatic treatment being

more reproducible [72] So-called helper or chaperone

probes target a sequence region adjacent to a probe target

site and have been demonstrated to increase target

accessibility by relieving secondary structures [5873]

However the helper probe approach is less practical for

high-density microarrays as each probe on the microarray

requires its own specific helper counterpart

Unique to formats involving the simultaneous hybrid-

ization of many probes is that the probes have to be fine-

tuned for uniform thermodynamic behaviour One way to

achieve this is by using probes that are identical in length

and adding tertiary amine salts such as tetramethylammo-

nium chloride to the hybridization andor washing buffers

[1556] Thereby differences in the G+C contents and

thus duplex stabilities among the probes are attenuated

[7475] Another strategy to equalize melting properties of

different oligonucleotide probes is to manipulate their

length [139]

One must realize that current in silico approaches for

predicting the hybridization behaviour of microarray

probes are limited due to the aforementioned reasons At

best they will lead to a pre-filtered set of candidate probes

whose true experimental performance will only be uncov-

ered after extensive empirical testing (Fig 2) In practice a

suitable set of test targets should contain at least one

perfectly matched target for each probe on the microarray

After challenging the microarrayrsquos performance by indi-

vidual hybridizations with each test target lsquolsquobadrsquorsquo probes

showing low sensitivity andor specificity are removed or

replaced Subsequently concentration series of targets


perfectly matching those probes that have displayed the

highest and lowest duplex yield should be hybridized to

the microarray giving an impression of the range of

sensitivities achievable for the individual probes In

conclusion the key to a reliable diagnostic microarray

lies within its concerted evaluation integrating in silico

predictions adjustment of hybridization conditions and

thorough testing in the laboratory

33 Further selected strategies to increase specificity and

or sensitivity

The most common way to enhance detectability of a target

gene is the use of PCR [1151776ndash78] PCR is applied to

focus the labelling to the target genes and thus to enrich them

relative to non-targeted background DNA In case of a single

marker gene highly selective PCR primers may achieve

optimal enrichment of target sequences from organisms of

interest [56] However a high number of selective PCR

primers is needed to cover all organisms targeted by a

microarray Running multiple PCR reactions considerably

increases the hands-on-time of the microarray assay and thus

simultaneous target amplification via multiplex PCR is

desirable [3579ndash82] Multiplex PCR has the inherent

drawback that potential biases increase with increased

complexity of the reactants (primerstarget genes) [the

number of successfully applied primer pairs within a multi-

plex PCR is normally low up to 12 [83]] Consequently

comparable to the actual microarray hybridization the

implementation of a pre-hybridization amplification step

based on multiplex PCR requires extensive careful testing

and validation

A promising approach to increase the sensitivity of a

microarray assay is tyramide signal amplification (TSA)

[51] Upon hybridization this method relies on enzymatic

amplification of the signal by employing horse radish

peroxidase-mediated deposition of fluorochrome-labelled

tyramides at the location of the probe

The ultimate specificity of microarray technology

depends on the discrimination between a fully complemen-

tary target and a non-target differing in only a single

nucleotide Recently various enzyme-assisted hybridization

strategies (also used in single nucleotide polymorphism and

resequencing assays [8485]) have gained attention because

of their promise in strongly discriminating single mis-

matches located near the 3V end of microarray probes

[2186ndash90]


For microarrays used in transcriptome analysis signal

intensity ratios indicating relative levels of expression are

obtained upon competitive hybridization of a control

sample versus the sample of interest (two color experi-

ment) [91] In contrast diagnostic microarray experiments

usually involve the hybridization of a single sample per

microarray (single color experiment) Fluorescence signals

for the individual probes are typically rated as present or

absent (Fig 1) a decision made depending on either

simple visual inspection of the scanned microarray image

or an arbitrary signal intensity threshold [11521

225880] Subsequently complex hybridization patterns

are in most cases still translated manually into inventory

lists of organisms present in the analyzed samples This is

a tedious procedure which becomes more problematic with

increasing numbers of probes per microarray and increas-

ing numbers of samples to be analyzed and thus

constitutes a major bottleneck of microarray-based diag-

nostics In an attempt to bridge the gap between data

collection and analysis a simple command line-based

program was recently developed for diagnostic microarrays

[15] [an advanced graphical user interface-based version

ChipAnalyser is under development (Harald Meier

unpublished data)]

Although systematic and stochastic errors associated

with the different steps during microarray fabrication and

target preparation greatly hamper quantification the funda-

mental potential of microarrays to provide quantitative data

on the abundance of the detected target in a sample is widely

acknowledged Adopting the two color hybridization

approach from transcriptome microarray analysis relative

abundance of targets in a sample (labelled in color one) can

be measured by competitive hybridization with target

mixtures of known concentrations (labelled in color two)

on the microarray [192] For single color hybridizations a

linear correlation between the signal intensity of a probe and

the concentration of the respective target sequence has been

observed for a certain range of target concentrations

[414793] However the slope of this linear relationship

will vary among the different probes immobilized on a

microarray because as mentioned previously different

probes display different affinities to their targets For a

reliable quantitative interpretation of a microarray hybrid-

ization a standard curve is thus needed for each probe on

the microarray This tedious evaluation procedure has not

been accomplished for microarrays published to date and

therefore the ability to correctly quantify across all probes

on a microarray is yet to be demonstrated A semi-

quantitative comparison of similar community structures

is however possible enabling the researcher to detect

spatial and temporal changes in microbial community

composition [39]


MDMs are increasingly used in clinical environmental

and food microbiology as well as in biowarfare agent

detection [43] (Table 1) While some clinical applications

are limited to genotyping of isolated strains there are more

and more publications on cultivation-independent rapid


detection and identification (in some cases also genotyping)

of pathogens from clinical specimens Microarrays used

merely for genotyping of isolates are outside the scope of

our review and are thus not listed in Table 1


Technologies used for MDMs are rather diverse PCR

amplification followed by conventional fluorescence label-

ling is used in most cases Specificity is then defined by the

hybridization step Depending on the marker genes applied

current MDMs can provide resolution at various taxonomic

levels down to species or even strain level Ribosomal RNA

genes are often used as marker genes but less so in clinical

microbiology due to their limited phylogenetic resolution

Alternative higher resolution phylogenetic markers are thus

beginning to be applied with short oligonucleotide probes

used in a hierarchical and parallel manner being the

preferred choice

Reliable analysis of 10ndash30 samples can be achieved

within 24 h (in most cases within a working day) by one

researcher The costs of consumables are approximately

30ndash100 EUR per analysis depending on the exact

methodology used Although this may seem expensive it

must be remembered that a single analysis provides parallel

detection and identification of tens to hundreds of micro-

organisms potentially present In addition basic genotyping

information can be obtained as well as semi-quantitative

information on the relative abundance of the detected

microorganisms The latter aspect is especially useful when

comparing similar samples for changes in the microbial

community structure (eg gut microbiota) Absolute

quantification is troubled by biases associated with for

example PCR amplification nucleic acid recovery and

different numbers of (marker) gene copies per genome The

sensitivity of MDMs is normally limited by the relative

abundance of the microbial population within the targeted

community with reported detection limits being 1ndash5

The hybridization potential of the probes is predicted based

on the nearest neighbour model and a number of empirical

microarray-specific factors

MDM technology lends itself to automation DNARNA

purification amplification labelling can all be carried out

by existing laboratory robots Moreover there are proof-of-

concept experiments where all these tasks are carried out in

a single lab-on-a-chip device [116] The subsequent hybrid-

ization wash and scan steps can also already be carried out

by various instruments in an automated manner (for details

please refer to httpgenomicshomecom) Automated

result interpretation is primarily a bioinformatics challenge

In general MDM technology is analogous to the applica-

tion of microarrays in gene expression profiling a high-

throughput screening tool with limited quantification

potential MDM results should thus be confirmed via

established lower-throughput but more sensitive andor

truly quantitative alternative methods such as fluorescence

in situ hybridization quantitative PCR or immunological

detection techniques

Even though MDMs are already widely applied in most

fields of microbiology anticipated improvements (listed and

discussed below) should overcome some of the aforemen-

tioned limitations

The prediction of the hybridization behaviour of the

probes is still rudimentary and requires improvement This

can be achieved via input from bioinformatics and the

establishment of datasets on array-specific effects such as

immobilisation and steric hindrance

Large databases comparable in size to that of the 16S

rRNA databases are critically needed for high resolution

phylogenetic markers Such markers will drastically

improve the applicability of MDMs for clinical and food

microbiology epidemiology and related fields If in such

databases sequence information is linked to clinical traits

(eg pathogenicity host specificity antibiotic resistance

geographic origin etc) MDM-based detection will serve

the additional purpose of providing a prediction of these

functions at least on some of them at a given level of

certainty

Some enzyme-based labelling methods [86ndash88117]

have demonstrated the potential for an improved detection

sensitivity (down to about 01 of the total community

targeted) although at the cost of losing all quantification

potential

Current hybridization platforms predominantly use

fluorescence-based optical detection Both the fluorescent

label and the detection devices confer high costs to this

technology seriously limiting its spread across diagnostic

laboratories With a few exceptions hybridization results

are read after irreversible processing steps thus the result

reflects hybridization under a selected suboptimal hybrid-

ization condition Alternative detection platforms are thus

being developed and elaborated which if successful could

have two major advantages One is reduced costs the other

is on-line detection of hybridization events Electronic and

mechanical detection methods [116118ndash121] require

cheaper or no labelling and simpler cheaper readout

devices Label-free detection may also help in cutting the

time-to-result a critical issue in many medical applica-

tions On-line detection of the hybridization event may be

possible via a number of techniques [8ndash10116118ndash125]

Such an improvement will drastically increase the infor-

mation obtainable from any hybridization event Associa-

tion and dissociation kinetics and maximum hybridization

signal at the optimal hybridization stringency for each

individual probe can all be obtained By considering these

extra information the reliability of MDM-based diagnos-

tics can be further improved On-line hybridization

detection is also another means of cutting the time-to-

result A label-free on-line hybridization detection

approach could make it possible to obtain results within

less than 2 h


Multiple marker genes providing reliable information on

the phylogeny and identity of the detected microbe and its

clinical traits should be used on a single MDM in many

cases to provide all the relevant information to the diagnostic

laboratory As discussed earlier multiplex amplification is

still a serious limitation to this approach A true on-chip PCR

where also the amplification primers are immobilised would

enable unlimited multiplexing solving this problem and

opening new horizons in the field

Acknowledgements

Related work at Seibersdorf research (LB) was funded by

the Fonds zur Forderung der wissenschaftlichen Forschung

Austria (P15044) and through the EU 5th Framework

Quality of Life and Management of Living Resources Grant

QLK-3 CT-2000-01528 Research of AL was funded by a

Marie Curie Intra-European Fellowship within the 6th

European Community Framework Programme Funding

from the EU towards COST Action 853 contributed by

enhancing exchange of ideas The authors thank Michael

Taylor for critical revision of the manuscript AL is greatly

indebted to Michael Wagner for ongoing support LB is

indebted to Fodor Szilvia for her support and fresh

independent views and ideas Helpful comments and

discussions from many active members of the Yahoo

discussion groups on microarrays and microbial diagnos-

ticsmicrobial ecology (httpgroupsyahoocomgroup

microarray and httpgroupsyahoocomgroupMDME)

are acknowledged

References

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microbial microarray for methanotrophs Environ Microbiol 20035

566ndash82

[2] Zhou J Microarrays for bacterial detection and microbial community

analysis Curr Opin Microbiol 20036288ndash94

[3] Letowski J Brousseau R Masson L DNA microarray applications in

environmental microbiology Anal Lett 2003363165ndash84

[4] Stenger D Andreadis J Voraa G Pancrazio J Potential applications

of DNA microarrays in biodefense-related diagnostics Curr Opin

Biotechnol 200213208ndash12

[5] Clewley JP A role for arrays in clinical virology fact or fiction J

Clin Virol 2004292ndash12

[6] Gershon D Microarrays go mainstream Nature Methods 20041

263ndash70

[7] Guschin DY Mobarry BK Proudnikov D Stahl DA Rittmann BE

Mirzabekov AD Oligonucleotide microchips as genosensors for

determinative and environmental studies in microbiology Appl

Environ Microbiol 1997632397ndash402

[8] Fotin AV Drobyshev AL Proudnikov DY Perov AN Mirzabekov

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[9] Anthony RM Schuitema ARJ Oskam L Klatser PR Direct

detection of Staphylococcus aureus mRNA using a flow through

microarray J Microbiol Methods 20056047ndash54

[10] Wu Y de Kievit P Vahlkamp L et al Quantitative assessment of a

novel flow-through porous microarray for the rapid analysis of gene

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[11] Urakawa H Noble PA El Fantroussi S Kelly JJ Stahl DA Single-

base-pair discrimination of terminal mismatches by using oligonu-

cleotide microarrays and neural network analyses Appl Environ

Microbiol 200268235ndash44

[12] Urakawa H El Fantroussi S Smidt H et al Optimization of single-

base-pair mismatch discrimination in oligonucleotide microarrays

Appl Environ Microbiol 2003692848ndash56

[13] Bodrossy L Sessitsch A Oligonucleotide microarrays in microbial

diagnostics Curr Opin Microbiol 20047245ndash54

[14] Ludwig W Schleifer KH Phylogeny of bacteria beyond the 16S

rRNA standard ASM News 1999651515ndash21

[15] Loy A Lehner A Lee N et al Oligonucleotide microarray for 16S

rRNA gene-based detection of all recognized lineages of sulfate-

reducing prokaryotes in the environment Appl Environ Microbiol

2002685064ndash81

[16] Loy A Horn M Wagner M probeBase an online resource for

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[17] Wilson KH Wilson WJ Radosevich JL et al High-density micro-

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Simultaneous discrimination between 15 fish pathogens by using 16S

ribosomal DNA PCR and DNA microarrays Appl Environ Micro-


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[21] Mitterer G Huber M Leidinger E et al Microarray-based

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amplification of 23S ribosomal DNA sequences J Clin Microbiol

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[22] Nubel U Schmidt PM Reiss E Bier F Beyer W Naumann D

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Microbiol Lett 2004240215ndash23

[23] Dahllof I Baillie H Kjelleberg S rpoB-based microbial community

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[24] Drancourt M Roux V Fournier PE Raoult D rpoB gene sequence-

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amplified polymorphic DNA gyrA and parC genes sequencing

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915ndash24

[27] Kakinuma K Fukushima M Kawaguchi R Detection and identi-

fication of Escherichia coli Shigella and Salmonella by microarrays

using the gyrB gene Biotechnol Bioeng 200383721ndash8

[28] Lloyd AT Sharp PM Evolution of the recA gene and the molecular

phylogeny of bacteria J Mol Evol 199337399ndash407

[29] Baldauf SL Palmer JD Doolittle WF The root of the universal tree

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[30] Ludwig W Neumaier J Klugbauer N et al Phylogenetic relation-

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Van Leeuwenhoek 199364285ndash305

[31] Wong RSY Chow AW Identification of enteric pathogens by heat

shock protein 60 kDa (HSP60) gene sequences FEMS Microbiol

Lett 2002206107ndash13

[32] Wertz JE Goldstone C Gordon DM Riley MA A molecular

phylogeny of enteric bacteria and implications for a bacterial species

concept J Evol Biol 2003161236ndash48

[33] Zwieb C Gorodkin J Knudsen B Burks J Wower J tmRDB

(tmRNA database) Nucleic Acids Res 200331446ndash7

[34] Sergeev N Volokhov D Chizhikov V Rasooly A Simultaneous

analysis of multiple staphylococcal enterotoxin genes by an

oligonucleotide microarray assay J Clin Microbiol 200442

2134ndash43

[35] Chizhikov V Rasooly A Chumakov K Levy DD Microarray

analysis of microbial virulence factors Appl Environ Microbiol

2001673258ndash63

[36] Volokhov D Rasooly A Chumakov K Chizhikov V Identification

of Listeria species by microarray-based assay J Clin Microbiol 2002

404720ndash8

[37] Saunders NA Underwood A Kearns AM Hallas G A virulence-

associated gene microarray a tool for investigation of the evolution

and pathogenic potential of Staphylococcus aureus Microbiology

20041503763ndash71

[38] Grimm V Ezaki S Susa M Knabbe C Schmid RD Bachmann

TT Use of DNA microarrays for rapid genotyping of TEM beta-

lactamases that confer resistance J Clin Microbiol 200442

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[39] Stralis-Pavese N Sessitsch A Weilharter A et al Optimisation of

diagnostic microarray for application in analysing landfill methano-

troph communities under different plant covers Environ Microbiol

20046347ndash63

[40] Rhee SK Liu X Wu L Chong SC Wan X Zhou J Detection of

genes involved in biodegradation and biotransformation in microbial

communities by using 50-mer oligonucleotide microarrays Appl


[41] Taroncher-Oldenburg G Griner EM Francis CA Ward BB

Oligonucleotide microarray for the study of functional gene diversity

in the nitrogen cycle in the environment Appl Environ Microbiol

2003691159ndash71

[42] Wu L Thompson DK Li G Hurt RA Tiedje JM Zhou J

Development and evaluation of functional gene arrays for detection

of selected genes in the environment Appl Environ Microbiol 2001

675780ndash90

[43] Hashsham SA Wick LM Rouillard JM Gulari E Tiedje JM

Potential of DNA microarrays for developing parallel detection tools

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research needs Biosens Bioelectron 200420668ndash83

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[45] Hughes TR Mao M Jones AR et al Expression profiling using

microarrays fabricated by an ink-jet oligonucleotide synthesizer Nat


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Assessment of the sensitivity and specificity of oligonucleotide

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[47] Tiquia SM Wu L Chong SC et al Evaluation of 50-mer

oligonucleotide arrays for detecting microbial populations in envi-

ronmental samples BioTechniques 200436664ndash5

[48] Letowski J Brousseau R Masson L Designing better probes effect

of probe size mismatch position and number on hybridization in

DNA oligonucleotide microarrays J Microbiol Methods 200457

269ndash78

[49] Vora GJ Meador CE Stenger DA Andreadis JD Nucleic acid

amplification strategies for DNA microarray-based pathogen detec-

tion Appl Environ Microbiol 2004703047ndash54

[50] Wang D Coscoy L Zylberberg M et al Microarray-based detection

and genotyping of viral pathogens Proc Natl Acad Sci U S A 2002

9915687ndash92

[51] Denef VJ Park J Rodrigues JL Tsoi TV Hashsham SA Tiedje JM

Validation of a more sensitive method for using spotted oligonucleo-

tide DNA microarrays for functional genomics studies on bacterial

communities Environ Microbiol 20035933ndash43

[52] Rouillard JM Zuker M Gulari E OligoArray 20 design of

oligonucleotide probes for DNA microarrays using a thermodynamic

approach Nucleic Acids Res 2003313057ndash62

[53] Gordon PM Sensen CW Osprey a comprehensive tool employing

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[54] Behr T Koob C Schedl M et al A nested array of rRNA targeted

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hybridization Syst Appl Microbiol 200023563ndash72

[55] Wagner M Horn M Daims H Fluorescence in situ hybridization for

the identification and characterisation of prokaryotes Curr Opin


[56] Loy A Schulz C Lucker S et al 16S rRNA gene-based

oligonucleotide microarray for environmental monitoring of the

betaproteobacterial order lsquolsquoRhodocyclalesrsquorsquo Appl Environ Microbiol

200571(3)1373ndash86

[57] Bodrossy L Diagnostic oligonucleotide microarrays for microbiol-

ogy In Blalock E editor A beginnerrsquos guide to microarrays 1 ed

New Yorkrsquo Kluwer Academic Publishers 2003 p 43ndash92

[58] Peplies J Lau SC Pernthaler J Amann R Glockner FO

Application and validation of DNA microarrays for the 16S

rRNA-based analysis of marine bacterioplankton Environ Microbiol

20046638ndash45

[59] Zuker M Mfold web server for nucleic acid folding and hybrid-

ization prediction Nucleic Acids Res 2003313406ndash15

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oligonucleotide DNA nearest-neighbor thermodynamics Proc Natl

Acad Sci U S A 1998951460ndash5

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buffer as a prerequisite for selection of 13mer microarray probe

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[62] Al Khaldi SF Myers KM Rasooly A Chizhikov V Genotyping of

Clostridium perfringens toxins using multiple oligonucleotide micro-

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Identification of Bacillus anthracis by multiprobe microarray hybrid-

ization Diagn Microbiol Infect Dis 200449163ndash71

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cleotide microchip for microbial identification studies a non-

equilibrium dissociation approach Environ Microbiol 20013

619ndash29

[65] Huang S Wang Y Chen P Qian HR Yeo A Bemis K Sum a new

way to incorporate mismatch probe measurements Genomics 2004

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[66] Lee I Dombkowski AA Athey BD Guidelines for incorporating

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influencing hybridization of nucleic acids to oligonucleotide arrays

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by universal amplification of 23S ribosomal DNA followed by

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land RF Atkins JF Thermodynamic calculations and statistical

correlations for oligo-probes design Nucleic Acids Res 200331

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prevents target hybridization to oligonucleotide microarrays Biosens

Bioelectron 200420728ndash35

[71] Southern EM DNA fingerprinting by hybridization to oligonucleo-

tide arrays Electrophoresis 1995161539ndash42

[72] Zhang XX Kosier B Priefer UB Genetic diversity of indigenous

Rhizobium leguminosarum bv viciae isolates nodulating two differ-

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[73] Chandler DP Newton GJ Small JA Daly DS Sequence versus

structure for the direct detection of 16S rRNA on planar

oligonucleotide microarrays Appl Environ Microbiol 200369

2950ndash8

[74] Maskos U Southern EM Parallel analysis of oligodeoxyribonucleo-

tide (oligonucleotide) interactions I Analysis of factors influencing

oligonucleotide duplex formation Nucleic Acids Res 199220

1675ndash8

[75] Jacobs KA Rudersdorf R Neill SD Dougherty JP Brown EL

Fritsch EF The thermal stability of oligonucleotide duplexes is

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cation to identifying recombinant DNA clones Nucleic Acids Res

1988164637ndash50

[76] Volokhov D Chizhikov V Chumakov K Rasooly A Microarray-

based identification of thermophilic Campylobacter jejuni C coli C

lari and C upsaliensis J Clin Microbiol 2003414071ndash80

[77] Bonch-Osmolovskaya EA Miroshnichenko ML Lebedinsky AV et

al Radioisotopic culture-based and oligonucleotide microchip

analyses of thermophilic microbial communities in a continental

high-temperature petroleum reservoir Appl Environ Microbiol 2003

696143ndash51

[78] Koizumi Y Kelly JJ Nakagawa T et al Parallel characterization of

anaerobic toluene-and ethylbenzene-degrading microbial consortia

by PCR-denaturing gradient gel electrophoresis RNA-DNA mem-

brane hybridization and DNA microarray technology Appl Environ


[79] Wang Z Vora GJ Stenger DA Detection and genotyping of

Entamoeba histolytica Entamoeba dispar Giardia lamblia and

Cryptosporidium parvum by oligonucleotide microarray J Clin


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for rapid detection and serotyping of acute respiratory disease-

associated adenoviruses J Clin Microbiol 2004423232ndash9

[81] Boriskin YS Rice PS Stabler RA et al DNA microarrays for virus

detection in cases of central nervous system infection J Clin


[82] Panicker G Call DR Krug MJ Bej AK Detection of pathogenic

vibrio spp in shellfish by using multiplex PCR and DNA micro-

arrays Appl Environ Microbiol 2004707436ndash44

[83] Rudi K Rud I Holck A A novel multiplex quantitative DNA array

based PCR (MQDA-PCR) for quantification of transgenic maize in

food and feed Nucleic Acids Res 200331e62

[84] Ericsson O Sivertsson A Lundeberg J Ahmadian A Microarray-

based resequencing by apyrase-mediated allele-specific extension

Electrophoresis 2003243330ndash8

[85] Lindroos K Sigurdsson S Johansson K Ronnblom L Syvanen AC

Multiplex SNP genotyping in pooled DNA samples by a four-color

microarray system Nucleic Acids Res 200230e70

[86] Rudi K Treimo J Nissen H Vegarud G Protocols for 16S rDNA

array analyses of microbial communities by sequence-specific label-

ing of DNA probes Sci World J 20033578ndash84

[87] Busti E Bordoni R Castiglioni B et al Bacterial discrimination by

means of a universal array approach mediated by LDR (ligase

detection reaction) BMC Microbiol 2002227

[88] Gharizadeh B Kaller M Nyren P et al Viral and microbial

genotyping by a combination of multiplex competitive hybridization

and specific extension followed by hybridization to generic tag

arrays Nucleic Acids Res 200331e146

[89] Lovmar L Fock C Espinoza F Bucardo F Syvanen AC Bondeson

K Microarrays for genotyping human group a rotavirus by multiplex

capture and type-specific primer extension J Clin Microbiol 2003

415153ndash8

[90] Mikhailovich V Lapa S Gryadunov D et al Identification of

rifampicin-resistant Mycobacterium tuberculosis strains by hybrid-

ization PCR and ligase detection reaction on oligonucleotide

microchips J Clin Microbiol 2001392531ndash40

[91] Schena M Microarray analysis Hoboken Canadarsquo John Wiley and

Sons Inc 2003

[92] Rudi K Treimo J Moen B Rud I Vegarud G Internal controls for

normalizing DNA arrays BioTechniques 33 (2002) 496 498 500

[93] Cho JC Tiedje JM Quantitative detection of microbial genes

by using DNA microarrays Appl Environ Microbiol 200268

1425ndash30

[94] Straub TM Daly DS Wunshel S Rochelle PA DeLeon R Chandler

DP Genotyping Cryptosporidium parvum with an hsp70 single-

nucleotide polymorphism microarray Appl Environ Microbiol 2002

681817ndash26

[95] Gonzalez SF Krug MJ Nielsen ME Santos Y Call DR Simulta-

neous detection of marine fish pathogens by using multiplex PCR

and a DNA microarray J Clin Microbiol 2004421414ndash9

[96] Keramas G Bang DD Lund M et al Use of culture PCR analysis

and DNA microarrays for detection of Campylobacter jejuni and

Campylobacter coli from chicken feces J Clin Microbiol 200442

3985ndash91

[97] Keramas G Bang DD Lund M et al Development of a sensitive

DNA microarray suitable for rapid detection of Campylobacter spp

Mol Cell Probes 200317187ndash96

[98] Castiglioni B Rizzi E Frosini A et al Development of a universal

microarray based on the ligation detection reaction and 16S rRNA

gene polymorphism to target diversity of cyanobacteria Appl


[99] Lehner A Loy A Behr T Gaenge H Schleifer K-H Wagner M

Oligonucleotide microarray for simultaneous identification and

differentiation of Enterococcus species in food samples FEMS

Microbiol Lett 2005246(1)133ndash42

[100] Sergeev N Distler M Courtney S et al Multipathogen oligonucleo-

tide microarray for environmental and biodefense applications

Biosens Bioelectron 200420684ndash98

[101] Peplies J Lau SCK Pernthaler J Amann R Glockner FO

Application and validation of DNA microarrays for the 16S rRNA-

based analysis of marine bacterioplankton Appl Environ Microbiol

200571(5)2723ndash31

[102] Fukushima M Kakinuma K Hayashi H Nagai H Ito K Kawaguchi

R Detection and identification of Mycobacterium species isolates by

DNA microarray J Clin Microbiol 2003412605ndash15

[103] Deng JY Zhang XE Lu HB et al Multiplex detection of mutations

in clinical isolates of rifampin-resistant Mycobacterium tuberculosis

by short oligonucleotide ligation assay on DNA chips J Clin


[104] Yu X Susa M Knabbe C Schmid RD Bachmann TT Development

and validation of a diagnostic DNA microarray to detect quinolone-

resistant Escherichia coli among clinical isolates J Clin Microbiol

2004424083ndash91

[105] Roth SB Jalava J Ruuskanen O Ruohola A Nikkari S Use of an

oligonucleotide array for laboratory diagnosis of bacteria responsible

for acute upper respiratory infections J Clin Microbiol 200442

4268ndash74

[106] Loy A Kusel K Lehner A Drake HL Wagner M Microarray and

functional gene analyses of sulfate-reducing prokaryotes in low-

sulfate acidic fens reveal cooccurrence of recognized genera and

novel lineages Appl Environ Microbiol 2004706998ndash7009

[107] Wilson WJ Strout CL DeSantis TZ Stilwell JL Carrano AV

Andersen GL Sequence-specific identification of 18 pathogenic

microorganisms using microarray technology Mol Cell Probes 2002

16119ndash27


[108] Chizhikov V Wagner M Ivshina A Hoshino Y Kapikian AZ

Chumakov K Detection and genotyping of human group a

rotaviruses by oligonucleotide microarray hybridization J Clin


[109] Klaassen CH Prinsen CF de Valk HA Horrevorts AM Jeunink

MA Thunnissen FB DNA microarray format for detection and

subtyping of human papillomavirus J Clin Microbiol 200442

2152ndash60

[110] Delrio-Lafreniere SA Browning MK McGlennen RC Low-density

addressable array for the detection and typing of the human

papillomavirus Diagn Microbiol Infect Dis 20044823ndash31

[111] Oh TJ Kim CJ Woo SK et al Development and clinical

evaluation of a highly sensitive DNA microarray for detection and

genotyping of human papillomaviruses J Clin Microbiol 200442

3272ndash80

[112] Sengupta S Onodera K Lai A Melcher U Molecular detection and

identification of influenza viruses by oligonucleotide microarray

hybridization J Clin Microbiol 2003414542ndash50

[113] Kessler N Ferraris O Palmer K Marsh W Steel A Use of the DNA

flow-thru chip a three-dimensional biochip for typing and subtyping

of influenza viruses J Clin Microbiol 2004422173ndash85

[114] Laassri M Chizhikov V Mikheev M Shchelkunov S Chumakov K

Detection and discrimination of orthopoxviruses using microarrays of

immobilized oligonucleotides J Virol Methods 200311267ndash78

[115] Lapa S Mikheev M Shchelkunov S et al Species-level identifica-

tion of orthopoxviruses with an oligonucleotide microchip J Clin


[116] Liu RH Yang J Lenigk R Bonanno J Grodzinski P Self-contained

fully integrated biochip for sample preparation polymerase chain

reaction amplification and DNA microarray detection Anal Chem

2004761824ndash31

[117] Baner J Isaksson A Waldenstrom E Jarvius J Landegren U Nilsson

M Parallel gene analysis with allele-specific padlock probes and tag

microarrays Nucleic Acids Res 200331e103

[118] McKendry R Zhang J Arntz Y et al Multiple label-free biodetection

and quantitative DNA-binding assays on a nanomechanical cantilever

array Proc Natl Acad Sci U S A 2002999783ndash8

[119] Gabig-Ciminska M Los M Holmgren A et al Detection of

bacteriophage infection and prophage induction in bacterial cultures

by means of electric DNA chips Anal Biochem 200432484ndash91

[120] Zimmermann K Eiter T Scheiflinger F Consecutive analysis of

bacterial PCR samples on a single electronic microarray J Microbiol

Methods 200355471ndash4

[121] Westin L Miller C Vollmer D et al Antimicrobial resistance and

bacterial identification utilizing a microelectronic chip array J Clin


[122] Meunier-Prest R Raveau S Finot E Legay G Cherkaoui-Malki M

Latruffe N Direct measurement of the melting temperature of

supported DNA by electrochemical method Nucleic Acids Res

200331e150

[123] Stimpson DI Hoijer JV Hsieh WT et al Real-time detection of

DNA hybridization and melting on oligonucleotide arrays by using

optical wave guides Proc Natl Acad Sci U S A 1995926379ndash83

[124] Nelson BP Liles MR Frederick KB Corn RM Goodman RM

Label-free detection of 16S ribosomal RNA hybridization on

reusable DNA arrays using surface plasmon resonance imaging


[125] Podsadlowski V Peter C Cammann K Brunen-Nieweler C

Borchers T Rapid species identification in food samples with an

evanescent-field-DNA-biosensor-chip 2004 Statusseminar Chip

Technologies Microarrays Hygiene and Health Frankfurt a Main

26-2712004

Fig 1 Typical steps of a diagnostic microarray experiment


categories according to their intended use Environmental

MDMs [23] are primarily applied in environmental and

industrial microbiology to obtain a picture of the structure of

the microbial community being analysed Requirements for

this class of MDMs are the parallel detection of many

microorganisms at the level of species genus or a higher

taxon and the potential for some level of quantification

Detectionidentification MDMs typically applied in clin-

ical veterinary food and biodefense microbiology [45]

must usually enable the reliable detection andor identifica-

tion at the speciessubspeciesstrain level of one or a few

microbes out of many that may be present in a sample It

Table 1



phylogenetic

resolution


samples

analysed








bacterial taxa






nirS nirK amoA nifH

pmoA dsrAB



sterility in mares









mixed cultures

[76]


intergenic spacer



swabs

[9697]




monella



ioplankton



















ALoyLBodrossy

Clin

icaChimica

Acta

363(2006)106ndash119

108


Escherichia coli



throat swabs

[105]





cyanobacterial



[15106]




formation waters

[77]


eukaryotes)



disease-associated

Adenoviruses


wash samples

[80]





(tonsillar cancer)

[111]






(varicella-zoster)





ALoyLBodrossy

Clin

icaChimica

Acta

363(2006)106ndash119

109














MDMs












in the near future






































































microarrays [43]






























[14751]














following section













procedures






















































[1153961]












Probe design






Refined probe set


Suitable software



Re-design


In silico

In vitro


Re-evaluation

12

34

ARB

ARB

ARBPrimRose



























12345678910111213141516171819202122232425262728

A1 A2 A3

B1 B2C1

D1

E1

A4



































































length [139]























or sensitivity




















and validation















[2186ndash90]

























unpublished data)]




























composition [39]
























preferred choice











































tioned limitations
















certainty





potential




























less than 2 h











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004

Table 1



phylogenetic

resolution


samples

analysed








bacterial taxa






nirS nirK amoA nifH

pmoA dsrAB



sterility in mares









mixed cultures

[76]


intergenic spacer



swabs

[9697]




monella



ioplankton



















ALoyLBodrossy

Clin

icaChimica

Acta

363(2006)106ndash119

108


Escherichia coli



throat swabs

[105]





cyanobacterial



[15106]




formation waters

[77]


eukaryotes)



disease-associated

Adenoviruses


wash samples

[80]





(tonsillar cancer)

[111]






(varicella-zoster)





ALoyLBodrossy

Clin

icaChimica

Acta

363(2006)106ndash119

109














MDMs












in the near future






































































microarrays [43]






























[14751]














following section













procedures






















































[1153961]












Probe design






Refined probe set


Suitable software



Re-design


In silico

In vitro


Re-evaluation

12

34

ARB

ARB

ARBPrimRose



























12345678910111213141516171819202122232425262728

A1 A2 A3

B1 B2C1

D1

E1

A4



































































length [139]























or sensitivity




















and validation















[2186ndash90]

























unpublished data)]




























composition [39]
























preferred choice











































tioned limitations
















certainty





potential




























less than 2 h











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004


Escherichia coli



throat swabs

[105]





cyanobacterial



[15106]




formation waters

[77]


eukaryotes)



disease-associated

Adenoviruses


wash samples

[80]





(tonsillar cancer)

[111]






(varicella-zoster)





ALoyLBodrossy

Clin

icaChimica

Acta

363(2006)106ndash119

109














MDMs












in the near future






































































microarrays [43]






























[14751]














following section













procedures






















































[1153961]












Probe design






Refined probe set


Suitable software



Re-design


In silico

In vitro


Re-evaluation

12

34

ARB

ARB

ARBPrimRose



























12345678910111213141516171819202122232425262728

A1 A2 A3

B1 B2C1

D1

E1

A4



































































length [139]























or sensitivity




















and validation















[2186ndash90]

























unpublished data)]




























composition [39]
























preferred choice











































tioned limitations
















certainty





potential




























less than 2 h











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004














MDMs












in the near future






































































microarrays [43]






























[14751]














following section













procedures






















































[1153961]












Probe design






Refined probe set


Suitable software



Re-design


In silico

In vitro


Re-evaluation

12

34

ARB

ARB

ARBPrimRose



























12345678910111213141516171819202122232425262728

A1 A2 A3

B1 B2C1

D1

E1

A4



































































length [139]























or sensitivity




















and validation















[2186ndash90]

























unpublished data)]




























composition [39]
























preferred choice











































tioned limitations
















certainty





potential




























less than 2 h











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004




















[14751]














following section













procedures






















































[1153961]












Probe design






Refined probe set


Suitable software



Re-design


In silico

In vitro


Re-evaluation

12

34

ARB

ARB

ARBPrimRose



























12345678910111213141516171819202122232425262728

A1 A2 A3

B1 B2C1

D1

E1

A4



































































length [139]























or sensitivity




















and validation















[2186ndash90]

























unpublished data)]




























composition [39]
























preferred choice











































tioned limitations
















certainty





potential




























less than 2 h











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004


Probe design






Refined probe set


Suitable software



Re-design


In silico

In vitro


Re-evaluation

12

34

ARB

ARB

ARBPrimRose



























12345678910111213141516171819202122232425262728

A1 A2 A3

B1 B2C1

D1

E1

A4



































































length [139]























or sensitivity




















and validation















[2186ndash90]

























unpublished data)]




























composition [39]
























preferred choice











































tioned limitations
















certainty





potential




























less than 2 h











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004

12345678910111213141516171819202122232425262728

A1 A2 A3

B1 B2C1

D1

E1

A4



































































length [139]























or sensitivity




















and validation















[2186ndash90]

























unpublished data)]




























composition [39]
























preferred choice











































tioned limitations
















certainty





potential




























less than 2 h











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004











or sensitivity




















and validation















[2186ndash90]

























unpublished data)]




























composition [39]
























preferred choice











































tioned limitations
















certainty





potential




























less than 2 h











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004


















preferred choice











































tioned limitations
















certainty





potential




























less than 2 h











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004











Acknowledgements


















are acknowledged

References




566ndash82











263ndash70




























2002685064ndash81



514ndash6













442ndash3




2004421048ndash57














1005ndash1011





915ndash24

























2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004















2134ndash43



2001673258ndash63



404720ndash8




20041503763ndash71




3766ndash3774




20046347ndash63








2003691159ndash71




675780ndash90







227ndash259













269ndash78






9915687ndash92




















200571(3)1373ndash86







20046638ndash45









29(1)1ndash10










619ndash29



84767ndash77




32681ndash90







38781ndash8




4211ndash7










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004










102297ndash305




2950ndash8




1675ndash8





1988164637ndash50








696143ndash51









































415153ndash8






Sons Inc 2003





1425ndash30




681817ndash26







3985ndash91


















200571(5)2723ndash31











2004424083ndash91




4268ndash74








16119ndash27









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004









2152ndash60







3272ndash80
















2004761824ndash31



















200331e150












26-2712004

Highly parallel microbial diagnostics using oligonucleotide microarrays

Documents

Transcript of Highly parallel microbial diagnostics using oligonucleotide microarrays